DESCRIPTION: Increased hepatic secretion of apolipoprotein B100 (apoB) is involved in the development of certain hyperlipidemias and atherosclerosis. Recent findings have demonstrated that hepatic apoB intracellular transport in the secretory pathway is a unique dynamic process that includes sorting of apoB molecules to secretion or degradation pathways. A candidate for the pathway that degrades apoB is the ubiquitin-proteasome system. The overall goal of this research is to understand the mechanism of hepatic apoB-lipoprotein secretion, including the precise pathway of intracellular transport of nascent apoB through the various compartments of the secretory pathway, and the pathway of degradation of apoB by the ubiquitin-proteasome system. The intracellular transport of endogenous and expressed apoB will be studied in HepG2 and McArdle Rh-7777 hepatoma cells. Studies will also be performed with primary pig hepatocytes to confirm observations made in cell lines and to study the physiological role of co- or post-translational apoB degradation. Aim I will investigate the location, orientation, and intracellular transport of nascent apoB in the endoplasmic reticulum (ER) and Golgi compartments of the secretory pathway using 1) puromycin-synchronized cells and immunocytochemistry of apoB and other proteins; 2) transport of apoB tagged with green fluorescent protein in live cells; and 3) nanogold immuno electron microscopy to more precisely localize apoB in or near organelles. Aim 2 will determine: 1) the mechanism of apoB transport to the proteasome; 2) the proteasome population (cytosol or ER-associated) involved in apoB degradation; 3) the role of the translocon in retrograde transport of apoB; and 4) the sites of ubiquitination in apoB. Aim 3 will take advantage of our newly developed permeabilized cell degradation assay for apoB and identify the protein(s) in rabbit reticulocyte lysate that stimulate the rapid proteasomal degradation of apoB. Significance: The entire secretory pathway of apoB will be elucidated in detail and proteins involved in the degradation of apoB will be characterized. A comprehensive understanding of the regulation of apoB secretion will shed light on hyperlipidemias involving overproduction of apoB-lipoproteins, possibly the result of inefficiency in the degradation of lipid-poor apoB by the proteasome pathway.